Six species of the genus Legionella, including L. pneumophila (the Legionnaire's Disease agent), contain, in addition to high levels of characteristic nonhydroxy fatty acids, significant levels of monohydroxy fatty acids and novel 2,3-dihydroxy fatty acids, a class of compounds without precedent in prokaryotes. The hydroxylated fatty acids are not extractable by common lipid solvents and remain bound to particulate cell matter, presumably cell surface macromolecules. An extensive literature has developed regarding the isolation and description of various cell surface molecules and their relationship to such biological activities of the genus Legionella as virulence (including the ability to survive and replicate in normally phagocytic cells), endotoxicity, and induction of various immune reactive substances (including antibodies, tumor necrosis factor, and interferon). However, in none of these reports has there been presented a satisfactory accounting of the presence, concentration, or relationship of the fatty acids to these macromolecules, suggesting that the fatty acids have not been sought, or when sought, have not been recognized. The objective of the proposed project is to determine the cellular location and linkage of the hydroxylated fatty acids with respect to cellular macromolecules. Cells of the six dihydroxy fatty acid- containing species will be fractionated according to well- documented procedures for obtaining cell surface components. All fractions, including those usually discarded in the normal application of the various procedures, will be examined for concentration and composition of the three fatty acid classes in relation to other parameters such as mass, total carbohydrate, total protein, and total phosphate. Fractions containing significant hydroxylated fatty acid levels will be further examined to obtain some insight into the type of moiety which bears the fatty acids and the linkage by which they are joined. Results of this study should increase our understanding of chemical structure and biological activities of this group of ubiquitous, opportunistically pathogenic bacteria.